Publication protocol
"pL0A_0–1, pL0A_0-R and pL0A_1-R Level 0 vectors [36] were linearized with HindIII as described in [36]. Inserts were amplified from templates using Phusion® High-Fidelity DNA Polymerase (NEB) and primers with flanking homologous sequences (S3 Table) according to manufacturer’s protocol. Following agarose gel electrophoresis, inserts were purified from the gel using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel) kits, assembled into Level 0 vectors using NEBuilder HiFi assembly master mix (NEB) and transformed into E. coli NEB 5α (NEB).
pL1A-hc / pL1A-lc (A0/AR) Level 1 vectors [36] were linearized with PacI (NEB). Expression cassettes were released from Level 0 vectors using PmeI (NEB), separated by agarose gel electrophoresis and purified from the gel as described above. Multiple cassettes were assembled into Level 1 vectors using TAR or NEBuilder HiFi. Plasmids were isolated from positive yeast colonies and transformed into electrocompetent ElectroMAX™DH5α-E™ Cells (Thermo Fisher Scientific). Multigene constructs were released from Level 1 vector with I-SceI (NEB). For inserts with similar length to the backbone, the plasmid was additionally digested with NheI (NEB) to allow separation of the insert and the backbone by gel electrophoresis. Following isolation from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), inserts were assembled into HindIII linearized Plant X-tender expression vectors by NEBuilder HiFi or SLiCE assembly method. Constructs were transformed into One Shot® TOP10 Chemically Competent E. coli (Thermo Fischer Scientific), homemade TOP10 chemically competent E. coli or homemade TOP10 electrocompetent E. coli. Incorrect assemblies were selected against by the expression of a suicide gene and by antibiotic selection."
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